Salmonella vaccine

ABSTRACT

The present invention relates to live attenuated  Salmonella  strains comprising a first attenuating mutation, that are not capable of making functional RecA. The invention also relates to these bacteria for use in vaccines. Furthermore, the invention relates to vaccines based upon these bacteria, to the use of such bacteria in the manufacture of vaccines and to methods for the preparation of such vaccines.

FIELD OF THE INVENTION

The present invention relates to an attenuated Salmonella straincomprising a first attenuating mutation, its use in vaccines, vaccinesbased upon this strain and methods for the preparation of such vaccines.

BACKGROUND OF THE INVENTION

Bacteria of the genus Salmonella are notorious for their pathogenicityin both man and animals. In the USA alone, on a yearly basis the numberof humans suffering from Salmonella infections exceeds the two millioncases. In most cases, the infection is caused by contaminated food.Well-known sources of infection are eggs (from both ducks and chickens),products containing eggs and not sufficiently heated poultry and pigmeat. Especially in infants, young children, elderly people and immunecompromised patients, the ability to cope with such infections is low.In these groups, the yearly death rate due to Salmonella infections ishigh. During the last few decades the more efficient large-scale animalhusbandry has led to an enormous increase in animal density. As aresult, an increase is seen of the number of animal infections andsubsequently in human infections caused by infected food. It is clearthat animals are the main source of Salmonella infection. This source isvery difficult to control. First of all Salmonella infections in mostcases cause no serious illness in healthy full-grown animals; theseanimals can carry the bacterium for a prolonged period. During that timethey are shedding the bacterium in their dung. This makes it practicallyimpossible to avoid infection in the more vulnerable young animals.Secondly, many Salmonella species colonise several different hostspecies. Some of the Salmonella species cause primary infections inspecific hosts, whereas other Salmonella species are not restrictive atall. As a primary infectans, S. typhi and paratyphi are frequentlyassociated with infection in man. S. dublin is connected with cattle, S.abortus-equi causes abortion in horses. S. abortus-ovi causes abortionin sheep. S. choleraesuis is the cause of lethal diarrhoea in youngpigs. S. typhimurium and S. enteritidis cause salmonellosis in humans,poultry, pigs, cattle and rodents, S. arizonae causes disease inturkeys, whereas S. gallinarum causes salmonellosis only in poultry.

It is clear that there is a need for good, safe and efficacious vaccinesfor combating the various Salmonella species. Currently, several liveattenuated Salmonella vaccines are commercially available.

Many of the strains that are suitable for use in a live vaccine areattenuated to a level that makes them virtually non-virulent. Twostriking examples of such non-virulent strains are the Salmonellagallinarum 9R strain and the Salmonella typhimurium SL3261 strain.

The Salmonella gallinarum 9R strain was described as long as 44 yearsago by Smith (J. Hyg. Camb., 54:419–432 (1956)). This highly attenuatedstrain is known to have at least a mutation in a gene involved in thesynthesis of the cell's O-polysaccharides, leading to a Smooth→Roughmutation. The 9R strain has been used as a Rough reference strain sincethen (see e.g. Cameron, C. M. et al. , Onderstepoort J. Vet. Res. 39(3),139–146 (1972)). This strain can be administered to chicken even in adose of 10⁹ bacteria without causing the death of any chicken, whereas adose comprising 10³ wild-type bacteria kills all animals.

The Salmonella typhimurium SL3261 strain has been described 19 years agoby Hoiseth, S. and Stocker, B. A. D. (Nature 291: 238–239(1981)) and isavailable from Deposit number SGSC 439, Salmonella Genetic Stock Centre,University of Calgary, Alberta, Canada T2N 1N4.

This strain is known to have a highly attenuating mutation in thearomatic pathway synthesis. This strain can be administered even in a10⁶ bacteria dose to susceptible mice without causing the death of anyanimal, whereas the LD₅₀ of the parent strain for these mice is lessthen 20 bacteria.

Such Salmonella strains have since long been appreciated for theirhighly attenuated character. They are so severely attenuated that theyare described in the literature as non-virulent. Therefore they havebeen the strains of choice for live attenuated Salmonella vaccines.

In principle, there is a clear relation between the level of virulenceand the level of immunity induced. In general the strains with thehighest virulence induce the highest levels of immunity: animals thatsurvive infections with wild-type Salmonella strains often build up along-lasting immunity. On the other hand, for use in vaccines thosestrains that induce no pathogenesis at all, the non-virulent strains,are the most desirable but such strains are often not capable ofinducing a sufficiently high level of immunity. The non-virulentSalmonella strains described above are just about capable of triggeringthe immune system to a sufficient level.

A relevant disadvantage of all live attenuated vaccines is however, thatin principle they can revert to a wild-type level of virulence throughrecombination of the mutated gene with DNA from bacteria that do carrynon-mutated genes, such as e.g. wild-type field strains. Such arecombination event can take place in various ways, e.g. throughtransfection, transduction or transformation. Genes that are known toplay a role in these recombination processes are known as rec-genes. Oneof the rec-genes of key importance is the recA-gene. This gene encodesan enzyme RecA that is involved in several steps of the recombinationprocess. recA and many other rec-genes have been described i.a. byLloyd. R. G. and Low, K. B. (“Escherichia coli and Salmonella”, sec.ed., ASM Press, ISBN 1-55581-0-845, par. 119 page 2236–2255), by West,S. C. (Annu. Rev. Biochem. 61: 603–640 (1992), and by Kowalczykowski. S.C. et al (Microbiol Rev. 58: 401–465 (1994)).

At first sight it seems therefore tempting, when contemplating a safelive attenuated vaccine, to delete one of the rec-genes. Such a deletionseverely impairs the ability of the bacterial DNA to recombine.Nevertheless, deletion of rec-genes is also known to cause severeattenuation of the bacterial strains from which it has been deleted.Deletion of e.g. the recA- or the recBC-genes in Salmonella makes themutants sensitive to the oxidative burst of macrophages, which leadsi.a. to significant in vivo growth suppression. This has beendemonstrated for Salmonella, e.g. by Buchmeier, N. A. et al., (Mol.Microbiol, 7:933–936 (1993)) and equally convincing for other bacterialgenera as distantly related to Salmonella as e.g. Vibrio cholerae(Ketley et al., Infect. and Immun. 58: 1481–1484 (1990)).

Thus, where deletion of a rec-gene might be an advantage for virulentstrains, it certainly would be assumed to be a disadvantage fornon-virulent strains, such as the Salmonella gallinarum 9R strain andthe Salmonella typhimurium SL3261 strain. These strains, known to bealready highly impaired, could not be expected to trigger anyimmunological response at all after removal of rec-genes. This mayexplain why no attempts were made to delete Rec-genes from such alreadyhighly attenuated, non-virulent strains as S. gallinarum 9R and S.typhimurium SL3261, in spite of their long history.

DETAILED DESCRIPTION OF THE INVENTION

It is an objective of the present invention to provide live attenuatedSalmonella vaccines that are both safe and efficacious. It wassurprisingly found now, that deletion of the virulence factor RecA doesnot further impair the ability of already attenuated Salmonella strainsto adequately trigger the immune system in comparison to theirRecA-positive counterparts.

Therefore, one embodiment of the invention provides attenuatedSalmonella strains that already comprise a first attenuating mutationand that have as a characteristic feature that they additionallycomprise a mutation that prohibits these strains from making afunctional RecA protein.

Attenuated strains that already comprise a first attenuating mutationare understood to be strains that have a sufficiently attenuatedcharacter to be used as a basis for live attenuated vaccines due to analready existing attenuation. The nature of that first attenuation isnot relevant. It can be any attenuating mutation or combination of twoor more mutations having a sufficiently attenuated character to make thebacterium useful as a basis for a live attenuated vaccine. Merely as anexample, it can e.g. be a Rough-mutation, a mutation in the histidin- orpurin synthesis pathway or a mutation in the aromatic amino acidbiosynthesis pathway. Also mutations in the cra-gene, andcrp/cya-mutations, known in the art are suitable mutations. Also, knownSalmonella vaccine strains are very suitable as a basis for theproduction of a RecA negative bacterium according to the presentinvention.

Strains not capable of making functional RecA are understood to bestrains that either make no RecA enzyme at all, or to a level that isessentially insufficient to lead to RecA-driven recombination events.This incapability to make RecA can i.a. be the result of mutation ordeletion of a part of, or of the whole recA gene, the recA promotor or agene involved in recA synthesis.

In a preferred form of this embodiment, Salmonella strains according tothe invention belong to the species Salmonella gallinarum or Salmonellatyphimurium.

In a more preferred form of this embodiment the Salmonella gallinarumstrain is Salmonella gallinarum 9R.

An even more preferred form of this embodiment relates to the strainSalmonella gallinarum 9R-RecA of which a sample is deposited with theCentraalbureau voor Schimmelcultures, Oosterstraat 1, 3742 SK Baarn, TheNetherlands under deposit number CBS 108964.

In another more preferred form of this embodiment the Salmonellatyphimurium strains is Salmonella typhimurium SL3261.

One possible way of making the recA gene or any of the other known genesinvolved in RecA-biosynthesis non-functional is by means of classicalmethods such as the treatment of wild-type bacteria with mutagenicagents such as base analogues, treatment with ultraviolet light ortemperature treatment (Anderson, P. 1995. Mutagenesis, p 31–58 inMethods in Cell Biology 48. H. F. Epstein and D. C. Shakes (Eds)). Othermethods for making RecA-negative mutants of various bacteria of whichthe wild type is RecA-positive, have been described i.a. by Liu, S. L.et al. (Infect. Immun. 1988 Aug; 56(8): 1967–73), Haas, R. et al., (Mol.Microbiol. 1993 May; 8(4): 753–60) and Graf, J. et al. (J. Bacteriol.1994 November; 176(22): 6986–91).

RecA-negative mutants can easily be selected because of their muchhigher sensitivity for U.V.-radiation. A classical way of detecting themutants amidst non-mutated bacteria is by replica-plating. Thistechnique starts with an agar plate covered with bacterial colonies thathave been subjected to a mutagenic treatment. A replica of this plate ismade onto a second agar plate. This plate is then subjected to a highdoes of U.V.-radiation. Those colonies that do no longer grow afterradiation treatment are colonies that have a mutation in a rec gene.Their live replica colonies on the original plate can then be selectedand grown for e.g. vaccine purposes.

The exact nature of the mutation caused by classical mutation techniquesis usually unknown. This can e.g. be a point mutation which may,although this is unlikely to happen, eventually revert to wild-type.Therefore transposon mutagenesis is a good alternative. Mutation bytransposon mutagenesis is also a mutagenesis-technique well-known in theart. This is a mutation accomplished at a localised site in thechromosome. A possibility to introduce a mutation at a predeterminedsite, rather deliberately than randomly, is offered by recombinantDNA-technology. Such a mutation may be an insertion, a deletion, areplacement of one nucleotide by another one or a combination thereof,with the only proviso that the mutated gene no longer encodes functionalRecA. Such a mutation can e.g. be made by deletion of a number of basepairs. Even very small deletions such as stretches of 10 base pairs canalready cause the recA-gene to encode a non-functional RecA. Even thedeletion of one single base pair may already lead to a non-functionalRecA, since as a result of such a mutation, the other base pairs are nolonger in the correct reading frame. Each deletion or insertion of anumber of base pairs indivisible by three causes such a frame shift.More preferably, a longer stretch is removed e.g. 100 base pairs. Evenmore preferably, the whole recA-gene is deleted.

All recombinant DNA techniques for the construction of recA-negativemutants are well-known standard techniques. They relate to cloning ofthe recA-gene, modification of the gene sequence by site-directedmutagenesis, restriction enzyme digestion followed by re-ligation orPCR-approaches and to subsequent replacement of the wild type recA genewith the mutant gene (allelic exchange or allelic replacement). Standardrecombinant DNA techniques such as cloning the recA gene in a plasmid,digestion of the gene with a restriction enzyme, followed byendonuclease treatment, re-ligation and homologous recombination in thehost strain, are all known in the art and described i.a. inManiatis/Sambrook (Sambrook, J. et al. Molecular cloning: a laboratorymanual. ISBN 0-87969-309-6). Site-directed mutations can e.g. be made bymeans of in vitro site directed mutagenesis using the Transformer® kitsold by Clontech. PCR-techniques are extensively described in(Dieffenbach & Dreksler; PCR primers, a laboratory manual. ISBN0-87969-447-5 (1995)).

Given the large amount of vaccines given nowadays to both pets and farmanimals, it is clear that combined administration of several vaccineswould be desirable, if only for reasons of decreased vaccination costs.It is therefore very attractive to use live attenuated bacteria as arecombinant carrier for heterologous genes, encoding antigens selectedfrom other pathogenic micro-organisms or viruses. Administration of sucha recombinant carrier has the advantage that immunity is induced againsttwo or more diseases at the same time. Live attenuated bacteria for usein a vaccine according to the present invention provide very suitablecarriers for heterologous genes. In principle such heterologous genescan be inserted in the bacterial genome at any non-essential site.

Therefore, a still even more preferred form of this embodiment relatesto bacteria according to the invention in which a heterologous gene isinserted. Such a heterologous gene can, as mentioned above, e.g. be agene encoding an antigen selected from other pathogenic micro-organismsor viruses. Such genes can e.g. be derived from pathogenic herpesviruses(e.g. the genes encoding the structural proteins of herpesviruses),Retroviruses (e.g. the gp160 envelope protein), adenoviruses and thelike. Also a heterologous gene can be obtained from pathogenic bacteria.As an example, genes encoding bacterial toxins such as Actinobacilluspleuropneumoniae toxins, Clostridium toxins, outer membrane proteins andthe like are very suitable bacterial heterologous genes. Further,heterologous genes from various parasites, such as e.g. Eimeria are veryattractive candidates for cloning in a live attenuated Salmonella vectoraccording to the invention. Another possibility is to insert a geneencoding a protein involved in triggering the immune system, such as aninterleukin, Tumor Necrosis Factor or an interferon, or another geneinvolved in immune-regulation.

In a most preferred form of this embodiment, the heterologous geneencodes an antigen of a micro-organism or virus that is selected fromthe group consisting of Infectious Bronchitis virus, Newcastle Diseasevirus, Infectious Bursal Disease (Gumboro), Chicken Anaemia agent, AvianReovirus, Mycoplasma gallisepticum, Turkey Rhinotracheitis virus,Haemophilus paragallinarum (Coryza), Chicken Poxvirus, AvianEncephalomyelitisvirus, Eimeria species, Pasteurella multocida,Mycoplasma synoviae, Salmonella species, Ornithobacterium rhinotrachealeand E. coli.

The use of the recA gene as an insertion site has the advantage thatthere is no need to find a new insertion site for the heterologous geneand at the same time the recA gene is inactivated and the newlyintroduced heterologous gene can be expressed (in concert with thehomologous bacterial genes). The construction of such recombinantcarriers can be done routinely, using standard molecular biologytechniques such as allelic exchange.

Thus, in a preferred form of this embodiment the heterologous gene isinserted in the recA gene. The heterologous gene can be insertedsomewhere in the recA gene or it can be inserted at the site of the recAgene while this gene has been partially or completely deleted.

Another embodiment of the invention relates to vaccines for combatingSalmonella infection that comprise an attenuated Salmonella strainaccording to the invention and a pharmaceutically acceptable carrier. Apharmaceutically acceptable carrier may be as simple as water, but itmay e.g. also comprise culture fluid in which the bacteria werecultured. Another suitable carrier is e.g. a solution of physiologicalsalt concentration.

The useful dosage to be administered will vary depending on the age,weight and animal vaccinated, the mode and route of administration andthe type of pathogen against which vaccination is sought. The vaccinemay comprise any dose of bacteria, sufficient to evoke an immuneresponse. Doses ranging between 10³ and 10¹⁰ bacteria are e.g. verysuitable doses. Doses between 10⁶ and 10⁹ bacteria are even morepreferred.

Optionally, one or more compounds having adjuvant activity may be addedto the vaccine. Adjuvants are non-specific stimulators of the immunesystem. They enhance the immune response of the host to the vaccine.Examples of adjuvants known in the art are Freunds Complete andIncomplete adjuvant, vitamin E, non-ionic block polymers,muramyldipeptides, ISCOMs (immune stimulating complexes, cf. forinstance European Patent EP 109942), Saponins, mineral oil, vegetableoil, and Carbopol. Adjuvants, specially suitable for mucosal applicationare e.g. the E. coli heat-labile toxin (LT) or Cholera toxin (CT). Othersuitable adjuvants are for example aluminium hydroxide, aluminiumphosphate or aluminium oxide, oil-emulsions (e.g. of Bayol F ^((R)) orMarcol 52 ^((R)), saponins or vitamin-E solubilisate.

Therefore, in a preferred form, the vaccines according to the presentinvention comprise an adjuvant.

Other examples of pharmaceutically acceptable carriers or diluentsuseful in the present invention include stabilisers such as SPGA,carbohydrates (e.g. sorbitol, mannitol, starch, sucrose, glucose,dextran), proteins such as albumin or casein, protein containing agentssuch as bovine serum or skimmed milk and buffers (e.g. phosphatebuffer). Especially when such stabilisers are added to the vaccine, thevaccine is very suitable for freeze-drying or spray-drying.

Therefore, in a more preferred form, the vaccine is in a freeze-dried orspray-dried form.

For administration to animals or humans, the vaccine according to thepresent invention can be given inter alia intranasally, by spraying,intradermally, subcutaneously, orally, by aerosol or intramuscularly.Preferred methods for reason of convenience are administration byspraying, intranasal administration and oral administration. Forapplication to poultry, wing-web and eye-drop administration areadditionally suitable.

Still another embodiment of the invention relates to attenuatedSalmonella strains according to the invention for use in a vaccine.

A further embodiment of the invention relates to attenuated Salmonellastrains according to the invention for use in the manufacture of avaccine for combating Salmonella infection.

The invention also relates to methods for the preparation of a vaccineaccording to the invention. Such methods comprise the admixing ofattenuated Salmonella bacteria according to the invention or antigenicmaterial thereof and a pharmaceutically acceptable carrier.

DEPOSIT OF BIOLOGICAL MATERIAL

Attenuated strain 9R ace was deposited on Sep. 21, 2000 under the termsof the Budapest Treaty at the Centraalbureau voor Schimmelcultures,Oosterstraat 1 P.O. Box 273, 3740 AG BAARN, The Netherlands, and hasbeen assigned Accession No. CBS 108964. Pursuant to 37 C.F.R. §1.808,the biological material deposited is made under two conditions. First,access to the deposit will be made available during pendency of thepatent application making reference to the deposit to one determined bythe Commissioner to be entitled thereto under 37 C.F.R. §1.14 and 35U.S.C. §122; and secondly with one exception, that restrictions imposedby the depositor on the availability to the public of the depositedmaterial be irrevocably removed upon the granting of the patent.

EXAMPLE 1

Construction of a recA Mutant of S. Gallinarum 9R and S. TyphimuriumSL3261.

The complete recA locus and flanking regions, approximately 4 kb, of S.gallinarum 9R and S. typhimurium SL3261 was cloned and sequenced.(Methods used for cloning were standard methods as described byManiatis/Sambrook (referenced above), RecA genes were detected asdescribed by Lloyd, West and Kowalczykowski (referenced above), methodsfor sequencing are standard methods well-known in the art). Based onthis sequence, the two flanking regions of the recA gene were amplifiedby PCR; 1 kb upstream recA and 1 kb downstream of recA. These two PCRfragments were connected to each other by overlap-extension PCR in sucha way that the orientation of the fragments was correct. This PCRfragment, representing the recA locus with a deletion of most of therecA gene, was cloned into a Salmonella suicide vector pLD55 (resistantto ampicillin and tetracycline) in Escherichia coli S17.1 lambda-pir.This strain was used for allelic exchange to make a clean, marker-freerecA mutant strain of S. gallinarum 9R and S. typhimurium SL3261, asdescribed below.

By means of conjugation the recA deletion construct in pLD55 wastransferred from E. coli into S. gallinarum 9R and S. typhimuriumSL3261. Since this plasmid does not replicate in Salmonella, ampicillinresistant S. gallinarum 9R or S. typhimurium SL3261 colonies had thedeletion construct integrated into their chromosome through homologousrecombination at the recA locus. This was checked by PCR using differentprimer sets. In a second, spontaneous, intrachromosomal recombinationstep between recA DNA regions, pLD55 and flanking DNA are excised whichlead to ampicillin and tetracycline sensitive colonies and at the sametime the formation of i) a wild type recA locus or ii) a mutated recAlocus containing the deletion as described above. Several of theampicillin and tetracycline sensitive colonies were analyzed for theirrecA locus by means of PCR: the wild type recA locus gave a fragment of1.4 kb and the deleted recA locus a fragment of 0.4 kb. For Salmonellagallinarum two out of 16 colonies showed the deleted recA locus and wereconfirmed to be recA mutants by sequence analysis of the recA PCRfragment and by their enhanced sensitivity to U.V. irradiation.

EXAMPLE 2

Vaccination Test

The efficacy against S. enteritidis of the 9R parent strain and the 9RrecA mutant CBS 108964 was tested in a challenge experiment inlaying-type chickens. Three groups of 15 previously unvaccinatedchickens were used. One group was vaccinated intramuscularly with the 9Rstrain (1.3×10⁷ CFU/dose), one group was vaccinated with the recA mutant(8.4×10⁶ CFU/dose) and the third group was used as control. All chickenswere challenge infected with a naladixic acid resistant S. enteritidisPT4 strain at 10 weeks of age (1.0×10⁸ CFU) via the oral route.

Two weeks after challenge, all chickens were necropsied and spleens,cloacal swabs and the caecum contents were cultured for the challengestrain. Direct inoculation on Brilliant Green Agar plates containingnaladixic acid (BGA) and plating after enrichment in buffered peptonewater containing naladixic acid was performed. Identity of S.enteritidis isolates was confirmed by agglutination with flagellumspecific antiserum.

Animals

Commercial layers, 6 weeks of age were obtained from a Salmonella freeflock.

Results

As shown in table 1, S. enteritidis colonisation rates of spleen andcloaca were highly reduced in both the 9R and the 9RrecA groups whencompared to unvaccinated controls. Therefore it can be concluded thatthe efficacy of vaccines based upon both 9R and the 9RrecA is fullycomparable.

TABLE 1 S. enteritidis positive Group 9R 9RrecA Control Spleen 1/15^(a)2/15^(a) 11/15^(b) Cloaca 0/15^(a) 0/15^(a) 9/15^(b) Caecum 2/15^(a,c)0/15^(a,b) 5/15^(c) Groups with different superscripts in a row differsignificantly (p < 0.05, Fisher exact test)

1. An attenuated Salmonella strain, wherein the strain: comprises a first attenuating mutation decreasing the LD₅₀ of said strain at least 50,000 times when compared to wild-type strain, comprises a mutation that prohibits the strain from making a functional RecA protein, and is a Salmonella gallinarum 9R strain.
 2. An attenuated Salmonella strain, wherein the strain is a Salmonella gallinarum 9R-RecA strain of which a sample is deposited with the Centraalbureau voor Schimmelcultures under deposit number CBS
 108964. 3. An attenuated Salmonella strain, wherein the strain: comprises a first attenuating mutation decreasing the LD₅₀ of said strain at least 50,000 times when compared to wild-type strain, comprises a mutation that prohibits the strain from making a functional RecA protein, and is a Salmonella typhimuriumSL3261 strain.
 4. A vaccine for combating Salmonella infection, comprising: an attenuated Salmonella strain according to claim 1, and a pharmaceutically acceptable carrier.
 5. A vaccine for combating Salmonella infection, comprising: an attenuated Salmonella strain according to claim 2, and a pharmaceutically acceptable carrier.
 6. A vaccine for combating Salmonella infection, comprising: an attenuated Salmonella strain according to claim 3, and a pharmaceutically acceptable carrier.
 7. A method for the preparation of a vaccine for combating Salmonella infection, comprising admixing: an attenuated Salmonella strain according to claim 1, and a pharmaceutically acceptable carrier.
 8. A method for the preparation of a vaccine for combating Salmonella infection, comprising admixing: an attenuated Salmonella strain according to claim 2, and a pharmaceutically acceptable carrier.
 9. A method for the preparation of a vaccine for combating Salmonella infection, comprising admixing: an attenuated Salmonella strain according to claim 3, and a pharmaceutically acceptable carrier. 